The interactions of serine proteases and protease nexins appear to play key roles in the regulation of cell growth and development in the tissues. Serine proteases are proteolytic enzymes having a reactive serine at the catalytic site. The presence of a nucleophilic group on the serine residue confers a general substrate specificity on this group of enzymes. Significant serine proteases comprise trypsin, chymotrypsin, thrombin, urokinase, plasmin, and elastase. Protease nexin-1 (PN-1) is a 45 kilodalton protein secreted by a variety of cultured cells, including human fibroblasts. PN-1 controls protease activity in the immediate environment of the cells, rapidly inhibiting the serine proteases, e.g., thrombin, urokinase and plasmin, by covalently binding to a serine group at the catalytic site. The complexes thus formed then bind back, by means of the PN portion of the complex, to the cells where they are internalized and degraded. This inhibition of protease enzymes modulates the response of the cell to the mitogenic effect of thrombin, and limits extracellular proteolysis and matrix degeneration mediated by urokinase (Cunningham, D. et al., J. Cellular Biochem. 32:281-291, 1986). The specificity of PN-1 is narrowed by binding to the extracellular matrix which accelerates its inhibition of thrombin and blocks its inhibition of urokinase and plasmin.
PN-1 is found to have a neurotrophic effect that appears to depend on its inhibition of thrombin. Neuroblastoma cells, as well as several types of primary neuronal cells in culture, rapidly extend neurites, (a morphological indication of differentiation) when switched from serum-containing to serum-free media. However, very low concentration of thrombin (2 nM) can cause cells in serum-free media to retract their neurites. It is presumably the presence of thrombin (and possibly other serine proteases) in serum-containing media that maintains neuroblastoma cells in a non-differentiated state.
In neural tissue, glial cells produce a neurite-stimulating factor which is a protease inhibitor with the same deduced amino acid sequence as PN-1. Both purified PN-1 and serum-free media conditioned by glioma cells promote neuroblastoma differentiation that can be blocked by added thrombin. Gurwitz, D., and D. Cunningham, Proc. Natl. Acad. Sci. USA, 85:3,440-3,444 (1988).
Recent studies on the brain tissues of patients with Alzheimer's disease indicate that an imbalance between proteases and protease nexin-1 may be involved in the etiology of the disease (Wagner, S. et al., Nature, submitted). The proposed pathological process implies that similar imbalances may occur in other neurological diseases, or when neural tissue is injured, so that abnormal amounts of serine protease are present in the tissues. Such imbalances could be corrected by providing PN-1 to complex these proteases so as to inhibit its repression of neural differentiation. However, present methods of isolation are not capable of producing PN-1 appropriately pure for these therapeutic uses.
Previously, PN-1 has been purified according to conventional techniques. One of these takes advantage of a heparin binding site on PN-1, and one step of the procedure involves fractionation over a heparin affinity resin or a resin with a similar affinity ligand, such as dextran sulfate. (Scott, R. and Baker, J. J. Bio. Chem. 258:10,439-10,444 (1983); Scott, R. et al., J. Bio. Chem. 260:7,029-7,034 (1985); and Farrell, D. et al., Biochem. J. 237:907-912 (1986)). During this purification step, other proteins with heparin binding sites can co-purify with PN-1 and contaminate the preparation.
It is therefore an object of the invention to provide highly specific monoclonal antibodies which can be used to purify PN-1 and so provide substantially pure protease inhibitor which is free of other biologically active molecules, and which can be a safe and effective agent for treating neurological diseases related to reduced levels of PN-1 in neural tissues.